Antisense oligonucleotide inhibition of raf gene expression

ABSTRACT

Oligonucleotides are provided which are targeted to nucleic acids encoding human c-raf and capable of inhibiting raf expression. The oligonucleotides contain a methoxyethoxy (2′—O—CH 2 CH 2 OCH 3 ) modification at the 2′ position of at least one nucleotide. Methods of inhibiting the expression of human raf using oligonucleotides of the invention are also provided. The present invention further comprises methods of inhibiting hyperproliferation of cells and methods of treating abnormal proliferative conditions which employ oligonucleotides of the invention.

This application is a continuation of U.S. application Ser. No.09/452,991 filed Dec. 2, 1999, now abandoned, which is a continuation ofU.S. application Ser. No. 09/064,792 filed Apr. 23, 1998, now abandoned,which is a continuation of U.S. application Ser. No. 08/463,912 filedJun. 5, 1995 now issued as U.S. Pat. No. 5,744,362, which is acontinuation-in-part of U.S. patent application Ser. No. 08/250,856,filed May 31, 1994, now issued as U.S. Pat. No. 5,563,255.

FIELD OF THE INVENTION

This invention relates to compositions and methods for modulatingexpression of the raf gene, a naturally present cellular gene which hasbeen implicated in abnormal cell proliferation and tumor formation. Thisinvention is also directed to methods for inhibiting hyperproliferationof cells; these methods can be used diagnostically or therapeutically.Furthermore, this invention is directed to treatment of conditionsassociated with expression of the raf gene.

BACKGROUND OF THE INVENTION

Alterations in the cellular genes which directly or indirectly controlcell growth and differentiation are considered to be the main cause ofcancer. The raf gene family includes three highly conserved genes termedA-, B- and c-raf (also called raf-1). Raf genes encode protein kinasesthat are thought to play important regulatory roles in signaltransduction processes that regulate cell proliferation. Expression ofthe c-raf protein is believed to play a role in abnormal cellproliferation since it has been reported that 60% of all lung carcinomacell lines express unusually high levels of c-raf mRNA and protein. Rappet al., The Oncogene Handbook, E. P. Reddy, A. M Skalka and T. Curran,eds., Elsevier Science Publishers, New York, 1988, pp. 213-253.

Oligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. For example, workers inthe field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases.

As examples, U.S. Pat. No. 5,135,917, issued Aug. 4, 1992, providesantisense oligonucleotides that inhibit human interleukin-1 receptorexpression. U.S. Pat. No. 5,098,890, issued Mar. 24, 1992 in the name ofGewirtz et al., is directed to antisense oligonucleotides complementaryto the c-myb oncogene and antisense oligonucleotide therapies forcertain cancerous conditions. U.S. Pat. No. 5,087,617, issued Feb. 11,1992, provides methods for treating cancer patients with antisenseoligonucleotides. U.S. Pat. No. 5,166,195 issued Nov. 24, 1992, providesoligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810, issued Apr.2, 1991, provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428,issued Mar. 16, 1993, provides antisense oligonucleotides havingantiviral activity against influenza virus. U.S. Pat. No. 4,806,463,issued Feb. 21, 1989, provides antisense oligonucleotides and methodsusing them to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717(Cohen et al.), issued Feb. 15, 1994, is directed to a mixed linkageoligonucleotide phosphorothioates complementary to an oncogene; U.S.Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 (Cohen et al.) aredirected to phosphorothioate oligonucleotide analogs used to preventreplication of foreign nucleic acids in cells. Antisenseoligonucleotides have been safely administered to humans and clinicaltrials of several antisense oligonucleotide drugs, targeted both toviral and cellular gene products, are presently underway. Thephosphorothioate oligonucleotide, ISIS 2922, has been shown to beeffective against cytomegalovirus retinitis in AIDS patients. BioWorldToday, Apr. 29, 1994, p. 3. It is thus established that oligonucleotidescan be useful therapeutic instrumentalities and can be configured to beuseful in treatment regimes for treatment of cells and animal subjects,especially humans.

Antisense oligonucleotide inhibition of gene expression has proven to bea useful tool in understanding the roles of raf genes. An antisenseoligonucleotide complementary to the first six codons of human c-raf hasbeen used to demonstrate that the mitogenic response of T cells tointerleukin-2 (IL-2) requires c-raf. Cells treated with theoligonucleotide showed a near-total loss of c-raf protein and asubstantial reduction in proliferative response to IL-2. Riedel et al.,Eur. J. Immunol. 1993, 23, 3146-3150. Rapp et al. have disclosedexpression vectors containing a raf gene in an antisense orientationdownstream of a promoter, and methods of inhibiting raf expression byexpressing an antisense Raf gene or a mutated Raf gene in a cell. WOapplication 93/04170. An antisense oligodeoxyribonucleotidecomplementary to codons 1-6 of murine c-Raf has been used to abolishinsulin stimulation of DNA synthesis in the rat hepatoma cell lineH4IIE. Tornkvist et al., J. Biol. Chem. 1994, 269, 13919-13921. WOApplication 93/06248 discloses methods for identifying an individual atincreased risk of developing cancer and for determining a prognosis andproper treatment of patients afflicted with cancer comprising amplifyinga region of the c-raf gene and analyzing it for evidence of mutation.

Denner et al. disclose antisense polynucleotides hybridizing to the genefor raf, and processes using them. WO 94/15645. Oligonucleotideshybridizing to human and rat raf sequences are disclosed.

Iversen et al. disclose heterotypic antisense oligonucleotidescomplementary to raf which are able to kill ras-activated cancer cells,and methods of killing raf-activated cancer cells. Numerousoligonucleotide sequences are disclosed, none of which are actuallyantisense oligonucleotide sequences.

There remains a long-felt need for improved compositions and methods forinhibiting raf gene expression.

SUMMARY OF THE INVENTION

The present invention provides oligonucleotides which are targeted tonucleic acids encoding human raf and are capable of inhibiting rafexpression. The oligonucleotides contain a methoxyethoxy(2′—O—CH₂CH₂OCH₃) modification at the 2′ position of at least onenucleotide. These oligonucleotides are believed to be useful bothdiagnostically and therapeutically, and are believed to be particularlyuseful in the methods of the present invention.

The present invention also comprises methods of inhibiting theexpression of human raf, particularly the abnormal expression of raf,using the oligonucleotides of the invention. These methods are believedto be useful both therapeutically and diagnostically as a consequence ofthe association between raf expression and hyperproliferation. Thesemethods are also useful as tools, for example for detecting anddetermining the role of raf expression in various cell functions andphysiological processes and conditions and for diagnosing conditionsassociated with raf expression.

The present invention also comprises methods of inhibitinghyperproliferation of cells using oligonucleotides of the invention.These methods are believed to be useful, for example in diagnosingraf-associated cell hyperproliferation. Methods of treating abnormalproliferative conditions are also provided. These methods employ theoligonucleotides of the invention. These methods are believed to beuseful both therapeutically and as clinical research and diagnostictools.

DETAILED DESCRIPTION OF THE INVENTION

Malignant tumors develop through a series of stepwise, progressivechanges that lead to the loss of growth control characteristic of cancercells, i.e., continuous unregulated proliferation, the ability to invadesurrounding tissues, and the ability to metastasize to different organsites. Carefully controlled in vitro studies have helped define thefactors that characterize the growth of normal and neoplastic cells andhave led to the identification of specific proteins that control cellgrowth and differentiation. The raf genes are members of a gene familywhich encode related proteins termed A-, B- and c-raf. Raf genes codefor highly conserved serine-threonine-specific protein kinases. Theseenzymes are differentially expressed; c-raf, the most thoroughlycharacterized, is expressed in all organs and in all cell lines thathave been examined. A- and B-raf are expressed in urogenital and braintissues, respectively. c-raf protein kinase activity and subcellulardistribution are regulated by mitogens via phosphorylation. Variousgrowth factors, including epidermal growth factor, acidic fibroblastgrowth factor, platelet-derived growth factor, insulin,granulocyte-macrophage colony-stimulating factor, interleukin-2,interleukin-3 and erythropoietin, have been shown to inducephosphorylation of c-raf. Thus, c-raf is believed to play a fundamentalrole in the normal cellular signal transduction pathway, coupling amultitude of growth factors to their net effect, cellular proliferation.

Certain abnormal proliferative conditions are believed to be associatedwith raf expression and are, therefore, believed to be responsive toinhibition of raf expression. Abnormally high levels of expression ofthe raf protein are also implicated in transformation and abnormal cellproliferation. These abnormal proliferative conditions are also believedto be responsive to inhibition of raf expression. Examples of abnormalproliferative conditions are hyperproliferative disorders such ascancers, tumors, hyperplasias, pulmonary fibrosis, angiogenesis,psoriasis, atherosclerosis and smooth muscle cell proliferation in theblood vessels, such as stenosis or restenosis following angioplasty. Thecellular signaling pathway of which raf is a part has also beenimplicated in inflammatory disorders characterized by T-cellproliferation (T-cell activation and growth), such as tissue graftrejection, endotoxin shock, and glomerular nephritis, for example.

It has now been found that elimination or reduction of raf geneexpression can halt or reverse abnormal cell proliferation. This hasbeen found even when levels of raf expression are not abnormally high.There is a great desire to provide compositions of matter which canmodulate the expression of the raf gene. It is greatly desired toprovide methods of detection of the raf gene in cells, tissues andanimals. It is also desired to provide methods of diagnosis andtreatment of abnormal proliferative conditions associated with abnormalraf gene expression. In addition, kits and reagents for detection andstudy of the raf gene are desired. “Abnormal” raf gene expression isdefined herein as abnormally high levels of expression of the rafprotein, or any level of raf expression in an abnormal proliferativecondition or state.

The present invention employs oligonucleotides targeted to nucleic acidsencoding raf. This relationship between an oligonucleotide and itscomplementary nucleic acid target to which it hybridizes is commonlyreferred to as “antisense”. “Targeting” an oligonucleotide to a chosennucleic acid target, in the context of this invention, is a multistepprocess. The process usually begins with identifying a nucleic acidsequence whose function is to be modulated. This may be, as examples, acellular gene (or mRNA made from the gene) whose expression isassociated with a particular disease state, or a foreign nucleic acidfrom an infectious agent. In the present invention, the target is anucleic acid encoding raf; in other words, the raf gene or mRNAexpressed from the raf gene. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe oligonucleotide interaction to occur such that the desiredeffect—modulation of gene expression—will result. Once the target siteor sites have been identified, oligonucleotides are chosen which aresufficiently complementary to the target, i.e., hybridize sufficientlywell and with sufficient specificity, to give the desired modulation.

In the context of this invention “modulation” means either inhibition orstimulation. Inhibition of raf gene expression is presently thepreferred form of modulation. This modulation can be measured in wayswhich are routine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application. “Hybridization”, in the context of this invention,means hydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which formtwo hydrogen bonds between them. “Specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe DNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted.

In the present invention, oligonucleotides are provided which aretargeted to mRNA encoding c-raf. In accordance with this invention,persons of ordinary skill in the art will understand that mRNA includesnot only the coding region which carries the information to encode aprotein using the three letter genetic code, but also associatedribonucleotides which form a region known to such persons as the5′-untranslated region, the 3′-untranslated region, the 5′ cap region,intron regions and intron/exon or splice junction ribonucleotides. Thus,oligonucleotides may be formulated which are targeted wholly or in partto these associated ribonucleotides as well as to the codingribonucleotides. The functions of messenger RNA to be interfered withinclude all vital functions such as translocation of the RNA to the sitefor protein translation, actual translation of protein from the RNA,splicing or maturation of the RNA and possibly even independentcatalytic activity which may be engaged in by the RNA. The overalleffect of such interference with the RNA function is to causeinterference with raf protein expression.

The present invention provides oligonucleotides for modulation of rafgene expression. Such oligonucleotides are targeted to nucleic acidsencoding c-raf.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term “oligonucleotide” also includes oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Modifications may be on one or more bases, sugars, orbackbone linkages, or combinations of these; such modifications are wellknown in the art. Modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The oligonucleotides may be chimeric oligonucleotides. “Chimericoligonucleotides” or “chimeras”, in the context of this invention, areoligonucleotides which contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the RNA target) and a region that is a substratefor RNase H cleavage. In one embodiment, a chimeric oligonucleotidecomprises at least one region modified to increase target bindingaffinity, and, usually, a region that acts as a substrate for RNAse H.Affinity of an oligonucleotide for its target (in this case a nucleicacid encoding raf) is routinely determined by measuring the Tm of anoligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate; dissociation is detectedspectrophotometrically. The higher the Tm, the greater the affinity ofthe oligonucleotide for the target.

Such modifications are routinely incorporated into oligonucleotides andthese oligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than 2′-deoxyoligonucleotides against a giventarget. The effect of such increased affinity is to greatly enhanceantisense oligonucleotide inhibition of raf gene expression. RNAse H isa cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of antisenseinhibition. Cleavage of the RNA target can be routinely demonstrated bygel electrophoresis. In another embodiment, the chimeric oligonucleotideis also modified to enhance nuclease resistance. Cells contain a varietyof exo- and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance. In somecases, oligonucleotide modifications which enhance target bindingaffinity are also, independently, able to enhance nuclease resistance.

The oligonucleotides of the present invention contain a methoxyethoxy(2′—O—CH₂CH₂OCH₃) modification at the 2′ position of at least onenucleotide. This modification has been shown to increase both affinityof the oligo nucleotide for its target and nuclease resistance of theoligonucleotide. Oligonucleotides in accordance with this invention arepreferably from about 8 to about 50 nucleotides in length. In thecontext of this invention it is understood that this encompassesnon-naturally occurring oligomers as hereinbefore described, having 8 to50 monomers.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Martin, P., Helv. Chim. Acta 1995, 78,486-504.Equipment for such synthesis is sold by several vendors includingApplied Biosystems. Any other means for such synthesis may also beemployed; the actual synthesis of the oligonucleotides is well withinthe talents of the routineer. It is also well known to use similartechniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other conjugated oligonucleotides.

It has now been found that certain oligonucleotides targeted to portionsof the c-raf mRNA are particularly useful for inhibiting raf expressionand for interfering with cell hyperproliferation. Methods for inhibitingc-raf expression using antisense oligonucleotides are, likewise, usefulfor interfering with cell hyperproliferation. In the methods of theinvention, tissues or cells are contacted with oligonucleotides. In thecontext of this invention, to “contact” tissues or cells with anoligonucleotide or oligonucleotides means to add the oligonucleotide(s),usually in a liquid carrier, to a cell suspension or tissue sample,either in vitro or ex vivo, or to administer the oligonucleotide(s) tocells or tissues within an animal.

For therapeutics, methods of inhibiting hyperproliferation of cells andmethods of treating abnormal proliferative conditions are provided. Theformulation of therapeutic compositions and their subsequentadministration is believed to be within the skill in the art. Ingeneral, for therapeutics, a patient suspected of needing such therapyis given an oligonucleotide in accordance with the invention, commonlyin a pharmaceutically acceptable carrier, in amounts and for periodswhich will vary depending upon the nature of the particular disease, itsseverity and the patient's overall condition. The pharmaceuticalcompositions of this invention may be administered in a number of waysdepending upon whether local or systemic treatment is desired, and uponthe area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal), oral, or parenteral, forexample by intravenous drip, intravenous injection or subcutaneous,intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

In addition to such pharmaceutical carriers, cationic lipids may beincluded in the formulation to facilitate oligonucleotide uptake. Onesuch composition shown to facilitate uptake is Lipofectin (BRL, BethesdaMD).

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a cure is effected or a diminution of diseasestate is achieved. Optimal dosing schedules can be calculated frommeasurements of drug accumulation in the body. Persons of ordinary skillcan easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual oligonucleotides, and can generally be calculatedbased on EC50's in in vitro and in vivo animal studies. For example,given the molecular weight of compound (derived from oligonucleotidesequence and chemical structure) and an effective dose such as an IC50,for example (derived experimentally), a dose in mg/kg is routinelycalculated.

The present invention is also suitable for diagnosing abnormalproliferative states in tissue or other samples from patients suspectedof having a hyperproliferative disease such as cancer, psoriasis orblood vessel restenosis or atherosclerosis. The ability of theoligonucleotides of the present invention to inhibit cell proliferationmay be employed to diagnose such states. A number of assays may beformulated employing the present invention, which assays will commonlycomprise contacting a tissue sample with an oligonucleotide of theinvention under conditions selected to permit detection and, usually,quantitation of such inhibition. Similarly, the present invention can beused to distinguish raf-associated tumors from tumors having otheretiologies, in order that an efficacious treatment regime can bedesigned.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

The oligonucleotides of the invention are also useful for detection anddiagnosis of raf expression. For example, radiolabeled oligonucleotidescan be prepared by 32p labeling at the 5′ end with polynucleotidekinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1989, Volume 2, p. 10.59. Radiolabeledoligonucleotides are then contacted with tissue or cell samplessuspected of raf expression and the sample is washed to remove unboundoligonucleotide. Radioactivity remaining in the sample indicates boundoligonucleotide (which in turn indicates the presence of raf) and can bequantitated using a scintillation counter or other routine means.Radiolabeled oligo can also be used to perform autoradiography oftissues to determine the localization, distribution and quantitation ofraf expression for research, diagnostic or therapeutic purposes. In suchstudies, tissue sections are treated with radiolabeled oligonucleotideand washed as described above, then exposed to photographic emulsionaccording to routine autoradiography procedures. The emulsion, whendeveloped, yields an image of silver grains over the regions expressingraf. Quantitation of the silver grains permits raf expression to bedetected.

Analogous assays for fluorescent detection of raf expression can bedeveloped using oligonucleotides of the invention which are conjugatedwith fluorescein or other fluorescent tag instead of radiolabeling. Suchconjugations are routinely accomplished during solid phase synthesisusing fluorescently labeled amidites or CPG (e.g., fluorescein-labeledamidites and CPG available from Glen Research, Sterling Va. See 1993Catalog of Products for DNA Research, Glen Research, Sterling Va., p.21).

Each of these assay formats is known in the art. One of skill couldeasily adapt these known assays for detection of raf expression inaccordance with the teachings of the invention providing a novel anduseful means to detect raf expression.

Inhibition of c-raf Expression by an Oligonucleotide Having SEQ ID NO: 1

Phosphorothioate deoxyoligonucleotides targeted to human c-raf weredesigned using the Genbank c-raf sequence HUMRAFR (Genbank listing×03484), synthesized and tested for inhibition of c-raf mRNA expressionin T24 bladder carcinoma cells using a Northern blot assay.Oligonucleotide ISIS 5132 (TCCCGCCTGTGACATGCATT; SEQ ID NO: 1), targetedto the 3′UTR of c-raf, showed greater than 90% inhibition.

Two oligonucleotides having SEQ ID NO: 1 and an 8-nucleotidedeoxynucleotide central region flanked on each side by 6 nucleotideshaving the 2′—O—CH₂CH₂OCH₃ modification were synthesized. Thesecompounds differ in that one of them, ISIS 10755 (also known as CIBA1440) has a uniform phosphorothioate backbone; the other, ISIS 10754(also known as CIBA 1439 or CGP 69845) has a phosphorothioate backbonein the central region (backbone linkages 7-14) and a phosphodiesterbackbone in the remaining (flanking) regions. These oligonucleotideswere tested for their ability to inhibit c-raf mRNA expression in T24cells. IC₅₀s were calculated (oligonucleotide concentration yielding 50%inhibition) and are shown in Table 1 along with Tm data showing affinityof these oligonucleotides for their complement. Because of theirextremely low IC₅₀s, both ISIS 10755 and ISIS 10754 are preferred.

TABLE 1 Antisense activity in T24 cells and Tm ISIS # Modification Tm (°C.) IC₅₀ (nM) SEQ ID NO: 5132 deoxy/P = S 62.2 125  1 107552′—O—CH₂CH₂OCH₃/ 76.1 20 1 P = S 10754 2′—O—CH₂CH₂OCH₃/ 77.5 20 1 P =S/P = O

Specificity of ISIS 5132 for Raf

Specificity of ISIS 5132 for raf mRNA was demonstrated by a Northernblot assay in which this oligonucleotide was tested for the ability toinhibit Ha-ras mRNA as well as c-raf mRNA in T24 cells. Ha-ras is acellular oncogene which is implicated in transformation andtumorigenesis. ISIS 5132 was shown to abolish c-raf MRNA almostcompletely with no effect on Ha-ras mRNA levels.

Chimeric Oligonucleotides

Chimeric oligonucleotides having SEQ ID NO: 1 were prepared. Theseoligonucleotides had central “gap” regions of 6, 8, or 10deoxynucleotides flanked by two regions of 2′-O-methyl modifiednucleotides. Backbones were uniformly phosphorothioate. In Northern blotanalysis, all three of these oligonucleotides (ISIS 6720, 6-deoxy gap;ISIS 6717, 8-deoxy gap; ISIS 6729, 10-deoxy gap) showed greater than 70%inhibition of c-raf mRNA expression in T24 cells. The 8-deoxy gapcompound (6717) showed greater than 90% inhibition. Oligonucleotides ofSEQ ID NO: 1 having an 8-deoxynucleotide gap flanked by either2′-O-propyl or 2′ fluoro modified nucleotides were also found to beactive.

Inhibition of Cancer Cell Proliferation

The phosphorothioate oligonucleotide ISIS 5132 (SEQ ID NO: 1) was shownto inhibit T24 bladder cancer cell proliferation. Cells were treatedwith various concentrations of oligonucleotide in conjunction withlipofectin (cationic lipid which increases uptake of oligonucleotide). Adose-dependent inhibition of cell proliferation was demonstrated, asindicated in Table 2, in which “None” indicates untreated control (nooligonucleotide) and “Control” indicates treatment with negative controloligonucleotide. Results are shown as percent inhibition compared tountreated control.

TABLE 2 Inhibition of T24 Cell Proliferation by ISIS 5132 Oligo conc.None Control 5132  50 nM 23% 0 +9% 100 nM 24% 0 +4% 250 nM 74% 0 10% 500nM 82% 0 18%

Effect of ISIS 5132 on T24 Human Bladder Carcinoma Tumors

Subcutaneous human T24 bladder carcinoma xenografts in nude mice wereestablished and treated with ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide administered intraperitoneally threetimes weekly at a dosage of 25 mg/kg. In this preliminary study, ISIS5132 inhibited tumor growth after eleven days by 35% compared tocontrols. Oligonucleotide-treated tumors remained smaller than controltumors throughout the course of the study.

Identification of Oligonucleotides Targeted to Rat and Mouse c-raf

Many conditions which are believed to be mediated by raf kinase are notamenable to study in humans. For example, tissue graft rejection is acondition which is likely to be ameliorated by interference with rafexpression; but, clearly, this must be evaluated in animals rather thanhuman transplant patients. Another such example is restenosis. Theseconditions can be tested in animal models, however, such as the rat andmouse models used here. These data provide added evidence of thetherapeutic utility of antisense oligonucleotides targeted to c-raf.

Oligonucleotide sequences for inhibiting c-raf expression in rat andmouse cells were identified. Rat and mouse c-raf genes have regions ofhigh homology; a series of oligonucleotides which target both rat andmouse c-raf mRNA sequence were designed and synthesized, usinginformation gained from evaluation of oligonucleotides targeted to humanc-raf. These oligonucleotides were screened for activity in mouse bENDcells and rat A-10 cells using Northern blot assays. Twooligonucleotides, ISIS 11061 and ISIS 10707, were found to inhibit c-rafRNA levels by greater than 90% in mouse bEND cells at a dose of 400 nM.These two oligonucleotides inhibited raf RNA levels virtually entirelyin rat A-10 cells at a concentration of 200 nM. The IC50 for ISIS 10707was found to be 170 nM in mouse bEND cells and 85 nM in rat A-10 cells.The IC50 for ISIS 11061 was determined to be 85 nM in mouse bEND cellsand 30 nM in rat A-10 cells.

Effect of ISIS 11061 on Endogenous c-raf mRNA Expression in Mice

Mice were injected intraperitoneally with ISIS 11061 (50 mg/kg) orcontrol oligonucleotide or saline control once daily for three days.Animals were sacrificed and organs were analyzed for c-raf mRNAexpression by Northern blot analysis. ISIS 11061 was found to decreaselevels of c-raf mRNA in liver by approximately 70%. Controloligonucleotides had no effects on c-raf expression. The effect of ISIS11061 was specific for c-raf; A-raf and G3PDH RNA levels were unaffectedby oligonucleotide treatment.

Antisense Oligonucleotide to c-raf Increases Survival in Murine HeartAllograft Model

To determine the therapeutic effects of the c-raf antisenseoligonucleotide ISIS 11061 in preventing allograft rejection, thisoligonucleotide was tested for activity in a murine vascularizedheterotopic heart transplant model. Hearts from C57BI10 mice weretransplanted into the abdominal cavity of C3H mice as primaryvascularized grafts essentially as described by Isobe et al.,Circulation 1991, 84, 1246-1255. Oligonucleotides were administered bycontinuous intravenous administration via a 7-day Alzet pump. The meanallograft survival time for untreated mice was 7.83+0.75 days(7, 7, 8,8, 8, 9 days). Allografts in mice treated for 7 days with 20 mg/kg or 40mg/kg ISIS 11061 all survived at least 11 days (11, 11, 12 days for 20mg/kg dose and >11, >11, >11 days for the 40 mg/kg dose).

In a pilot study conducted in rats, hearts from Lewis rats weretransplanted into the abdominal cavity of ACI rats. Rats were dosed withISIS 11061 at 20 mg/kg for 7 days via Alzet pump. The mean allograftsurvival time for untreated rats was 8.86+0.69 days (8, 8, 9, 9, 9, 9,10 days). In rats treated with oligonucleotide, the allograft survivaltime was 15.3±1.15 days (14, 16, 16 days).

Effects of Antisense Oligonucleotide Targeted to c-raf on Smooth MuscleCell Proliferation

Smooth muscle cell proliferation is a cause of blood vessel stenosis,for example in atherosclerosis and restenosis after angioplasty.Experiments were performed to determine the effect of ISIS 11061 onproliferation of A-10 rat smooth muscle cells. Cells in culture weregrown with and without ISIS 11061 (plus lipofectin) and cellproliferation was measured 24 and 48 hours after stimulation with fetalcalf serum. ISIS 11061 (500 nM) was found to inhibit serum-stimulatedcell growth in a dose-dependent manner with a maximal inhibition of 46%and 75% at 24 hours and 48 hours, respectively. An IC50 value of 200 nMwas obtained for this compound. An unrelated control oligonucleotide hadno effect at doses up to 500 nM.

Effects of Antisense Oligonucleotides Targeted to c-raf on Restenosis inRats

A rat carotid artery injury model of angioplasty restenosis has beendeveloped and has been used to evaluate the effects on restenosis ofantisense oligonucleotides targeted to the c-myc oncogene. Bennett etal., J. Clin. Invest. 1994, 93, 820-828. This model will be used toevaluate the effects of antisense oligonucleotides targeted to ratc-raf, particularly ISIS 11061, on restenosis. Following carotid arteryinjury with a balloon catheter, oligonucleotides are administered eitherby intravenous injection, continuous intravenous administration viaAlzet pump, or direct administration to the carotid artery in a pluronicgel matrix as described by Bennett et al. After recovery, rats aresacrificed, carotid arteries are examined by microscopy and effects oftreatment on luminal cross-sections are determined.

The invention is further illustrated by the following examples which areillustrations only and are not intended to limit the present inventionto specific embodiments.

EXAMPLES Example 1 Synthesis and Characterization of Oligonucleotides

Unmodified DNA oligonucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For hosphorothioate oligonucleotides,the standard oxidation ottle was replaced by a 0.2 M solution ofH-1,2-enzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.2′-O-methyl phosphorothioate oligonucleotides were synthesized using2′-O-methyl β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, NeedhamMass.) and the standard cycle for unmodified oligonucleotides, exceptthe wait step after pulse delivery of tetrazole and base was increasedto 360 seconds. The 3′-base used to start the synthesis was a2′-deoxyribonucleotide. 2′-O-propyl oligonucleotides were prepared by aslight modification of this procedure.

2′-fluoro phosphorothioate oligonucleotides were synthesized using5′-dimethoxytrityl-3′-phosphoramidites and prepared as disclosed in U.S.patent application Ser. No. 463,358, filed Jan. 11, 1990, and Ser. No.566,977, filed Aug. 13, 1990, which are assigned to the same assignee asthe instant application and which are incorporated by reference herein.The 2′-fluoro oligonucleotides were prepared using phosphoramiditechemistry and a slight modification of the standard DNA synthesisprotocol: deprotection was effected using methanolic ammonia at roomtemperature.

The methoxyethoxy (2′—Q—CH₂CH₂OCH₃) oligonucleotides were synthesizedaccording to the method of Martin, Helv. Chim. Acta 1995, 78,486-504.

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides were purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

Example 2 Northern Blot Analysis of Inhibition of c-raf mRNA Expression

The human urinary bladder cancer cell line T24 was obtained from theAmerican Type Culture Collection (Rockville Md.). Cells were grown inMcCoy's SA medium with L-glutamine (Gibco BRL, Gaithersburg Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEMreduced-serum medium containing 2.5 μl DOTMA. Oligonucleotide withlipofectin was then added to the desired concentration. After 4 hours oftreatment, the medium was replaced with McCoy's medium. Cells wereharvested 24 to 72 hours after oligonucleotide treatment and RNA wasisolated using a standard CsCl purification method. Kingston, R. E., inCurrent Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.),John Wiley and Sons, NY. Total RNA was isolated by centrifugation ofcell lysates over a CsCl cushion. RNA samples were electrophoresedthrough 1.2% agarose-formaldehyde gels and transferred to hybridizationmembranes by capillary diffusion over a 12-14 hour period. The RNA wascross-linked to the membrane by exposure to UV light in a Stratalinker(Stratagene, La Jolla, Calif.) and hybridized to random-primed³²P-labeled c-raf cDNA probe (obtained from ATCC) or G3PDH probe as acontrol. RNA was quantitated using a Phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

Example 3 Specific Inhibition of c-raf Kinase Protein Expression in T24Cells

T24 cells were treated with oligonucleotide (200 nM) and lipofectin atT=0 and T=24 hours. Protein extracts were prepared at T=48 hours,electrophoresed on acrylamide gels and analyzed by Western blot usingpolyclonal antibodies against c-raf (UBI, Lake Placid, N.Y.) or A-raf(Transduction Laboratories, Knoxville, Tenn.). Radiolabeled secondaryantibodies were used and raf protein was quantitated using aPhosphorimager (Molecular Dynamics, Sunnyvale Calif.).

Example 4 Antisense Inhibition of Cell Proliferation

T24 cells were treated on day 0 for two hours with variousconcentrations of oligonucleotide and lipofectin (50 nM oligonucleotidein the presence of 2 μg/ml lipofectin; 100 nM oligonucleotide and 2μg/ml lipofectin; 250 nM oligonucleotide and 6 μg/ml lipofectin or 500nM oligonucleotide and 10 μg/ml lipofectin). On day 1, cells weretreated for a second time at desired oligonucleotide concentration fortwo hours. On day 2, cells were counted.

Example 5 Effect of ISIS 5132 on T24 Human Bladder Carcinoma TumorXenografts in Nude Mice

5×10⁶ T24 cells were implanted subcutaneously in the right inner thighof nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) were administeredthree times weekly beginning on day 4 after tumor cell inoculation. Asaline-only control was also given. Oligonucleotides were given byintraperitoneal injection. Oligonucleotide dosage was 25 mg/kg. Tumorsize was measured and tumor volume was calculated on the eleventh,fifteenth and eighteenth treatment days.

Example 6 Effect of ISIS 5132 on MDA-MB 231 Human Breast Carcinoma TumorXenografts in Nude Mice

5×10⁶ MDA-MB 231 cells are implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) are administeredonce daily beginning on day 10 after tumor cell inoculation. Asaline-only control is also given. Oligonucleotides are given byintravenous injection at a dosage of 2 mg/kg to 25 mg/kg. Tumor size ismeasured and tumor volume is calculated on days 10, 13, 16, 20, 23 and27 following tumor cell inoculation.

For intraperitoneal oligonucleotide administration, oligonucleotides areadministered once daily beginning on day after tumor cell inoculation. Asaline-only control is also given. Oligonucleotides are given byintraperitoneal injection at a dosage of 2 mg/kg to 25 mg/kg. Tumor sizeis measured and tumor volume is calculated on days 10, 13, 16, 20, 23and 27 following tumor cell inoculation.

Example 7 Effect of ISIS 5132 on Colo 205 Human Colon Carcinoma TumorXenografts in Nude Mice

5×10⁶ Colo 205 cells are implanted subcutaneously in the right innerthigh of nude mice. Oligonucleotides (ISIS 5132 and an unrelated controlphosphorothioate oligonucleotide suspended in saline) are administeredonce per day beginning on day 5 after tumor cell inoculation. Asaline-only control is also given. Oligonucleotides are given byintravenous injection. Oligonucleotide dosage is 2 mg/kg to 25 mg/kg.Tumor size is measured and tumor volume is calculated on days 5, 8, 11,14, 18, 22 and 25 after tumor inoculation.

Example 8 Diagnostic Assay for Raf-associated Tumors Using Xenografts inNude Mice

Tumors arising from raf expression are diagnosed and distinguished fromother tumors using this assay. A biopsy sample of the tumor is treated,e.g., with collagenase or trypsin or other standard methods, todissociate the tumor mass. 5×10⁶ tumor cells are implantedsubcutaneously in the inner thighs of two or more nude mice. Antisenseoligonucleotide (e.g., ISIS 5132) suspended in saline is administered toone or more mice by intraperitoneal injection three times weeklybeginning on day 4 after tumor cell inoculation. Saline only is given toa control mouse. oligonucleotide dosage is 25 mg/kg. Tumor size ismeasured and tumor volume is calculated on the eleventh treatment day.Tumor volume of the oligonucleotide-treated mice is compared to that ofthe control mouse. The volume of raf-associated tumors in the treatedmice are measurably smaller than tumors in the control mouse. Tumorsarising from causes other than raf expression are not expected torespond to the oligonucleotides targeted to raf and, therefore, thetumor volumes of oligonucleotide-treated and control mice areequivalent.

Example 9 Detection of Raf Expression

Oligonucleotides are radiolabeled after synthesis by ³²P labeling at the5′ end with polynucleotide kinase. Sambrook et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2,pg. 11.31-11.32. Radiolabeled oligonucleotides are contacted with tissueor cell samples suspected of raf expression, such as tumor biopsysamples or skin samples where psoriasis is suspected, under conditionsin which specific hybridization can occur, and the sample is washed toremove unbound oligonucleotide. Radioactivity remaining in the sampleindicates bound oligonucleotide and is quantitated using a scintillationcounter or other routine means. Radiolabeled oligonucleotides of theinvention are also used in autoradiography. Tissue sections are treatedwith radiolabeled oligonucleotide and washed as described above, thenexposed to photographic emulsion according to standard autoradiographyprocedures. The emulsion, when developed, yields an image of silvergrains over the regions expressing raf. The extent of raf expression isdetermined by quantitation of the silver grains.

Analogous assays for fluorescent detection of raf expression useoligonucleotides of the invention which are labeled with fluorescein orother fluorescent tags. Labeled DNA oligonucleotides are synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). Fluorescein-labeled amidites arepurchased from Glen Research (Sterling Va.). Incubation ofoligonucleotide and biological sample is carried out as described forradiolabeled oligonucleotides except that instead of a scintillationcounter, a fluorimeter or fluorescence microscope is used to detect thefluorescence which indicates raf expression.

Example 10 A549 Xenografts

5×10⁶ A549 cells are implanted subcutaneously in the inner thigh of nudemice. Oligonucleotides (ISIS 5132 and CGP 69845, also known as ISIS10754) suspended in saline are administered once daily by intravenousinjection at doses ranging from 2 mg/kg to 25 mg/kg. Resulting tumorsare measured on days 9, 12, 17 and 21 and tumor volumes are calculated.

Example 11 Effect of Oligonucleotide on Endogenous c-raf Expression

Mice were treated by intraperitoneal injection at an oligonucleotidedose of 50 mg/kg on days 1, 2 and 3. On day 5 4, animals were sacrificedand organs removed for c-rat mRNA assay by Northern blot analysis. Fourgroups of animals were employed: 1) no oligonucleotide treatment(saline); 2) negative control oligonucleotide ISIS 1082 (targeted toherpes simplex virus; 3) negative control oligonucleotide 4189 (targetedto mouse protein kinase C-α; 4) ISIS 11061 targeted to rodent c-raf.

Example 12 Cardiac Allograft Rejection Model

Hearts were transplanted into the abdominal cavity of rats or mice (of adifferent strain from the donor) as primary vascularized graftsessentially as described by Isobe et al., Circulation 1991, 84,1246-1255. Oligonucleotides were administered by continuous intravenousadministration via a 7-day Alzet pump. Cardiac allograft survival wasmonitored by listening for the presence of a second heartbeat in theabdominal cavity.

Example 13 Proliferation Assay Using Rat A-10 Smooth Muscle Cells

A10 cells were plated into 96-well plates in Dulbecco's modified Eaglemedium (DMEM)+10% fetal calf serum and allowed to attach for 24 hours.Cells were made quiescent by the addition of DMEM+0.2% dialyzed fetalcalf serum for an additional 24 hours. During the last 4 hours ofquiescence, cells were treated with ISIS 11061 +lipofectin (Gibco-BRL,Bethesda Md.) in serum-free medium. Medium was then removed, replacedwith fresh medium and the cells were stimulated with 10% fetal calfserum. The plates were the placed into the incubator and cell growth wasevaluated by MTS conversion to formozan (Promega cell proliferation kit)at 24 and 48 hours after serum stimulation. A control oligonucleotide,ISIS 1082 (an unrelated oligonucleotide targeted to herpes simplexvirus), was also tested.

Example 14 Rat Carotid Artery Restenosis Model

This model has been described by Bennett et al., J. Clin. Invest. 1994,93, 820-828. Intimal hyperplasia is induced by balloon catheterdilatation of the carotid artery of the rat. Rats are anesthetized andcommon carotid artery injury is induced by passage of a balloonembolectomy catheter distended with 20 ml of saline. Oligonucleotidesare applied to the adventitial surface of the arterial wall in apluronic gel solution. Oligonucleotides are dissolved in a 0.25%pluronic gel solution at 4° C. (F127, BASF Corp.) at the desired dose.100 μl of the gel solution is applied to the distal third of the commoncarotid artery immediately after injury. Control rats are treatedsimilarly with gel containing control oligonucleotide or nooligonucleotide. The neck wounds are closed and the animals allowed torecover. 14 days later, rats are sacrificed, exsanguinated and thecarotid arteries fixed in situ by perfusion with paraformaldehyde andglutaraldehyde, excised and processed for microscopy. Cross-sections ofthe arteries are calculated.

In an alternative to the pluronic gel administration procedure, rats aretreated by intravenous injection or continuous intravenous infusion (viaAlzet pump) of oligonucleotide.

1 1 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic 1 tcccgcctgt gacatgcatt 20

What is claimed is:
 1. An antisense oligonucleotide up to 50 nucleotidesin length and comprising SEQ ID NO: 1, wherein said antisenseoligonucleotide inhibits human raf gene expression.
 2. A method ofinhibiting the expression of human raf comprising contacting humantissues or cells which express human raf with an antisenseoligonucleotide up to 50 nucleotides in length and comprising SEQ ID NO:1, wherein said antisense oligonucleotide inhibits human raf geneexpression.
 3. The method of claim 2 wherein said expression of humanraf is abnormal expression.
 4. A method of inhibiting hyperproliferationof human cells comprising contacting hyperproliferating human cells withan antisense oligonucleotide up to 50 nucleotides in length andcomprising SEQ ID NO: 1, wherein said antisense oligonucleotide inhibitshuman raf gene expression.
 5. A method of treating an abnormalproliferative condition comprising contacting a human or cells, tissuesor a bodily fluid of said human, suspected of having an abnormalproliferative condition with an antisense oligonucleotide up to 50nucleotides in length and comprising SEQ ID NO: 1, wherein saidantisense oligonucleotide inhibits human raf gene expression.
 6. Themethod of claim 5 wherein the condition is a hyperproliferativedisorder.
 7. A method of inhibiting proliferation of human cancer cellsin a subject comprising administering to the subject an antisenseoligonucleotide up to 50 nucleotides in length and comprising SEQ ID NO:1, wherein said antisense oligonucleotide further comprises at least onephosphorothioate internucleoside linkage or at least one 2′ modificationof the sugar, and wherein said antisense oligonucleotide inhibits humanraf gene expression.
 8. A method of increasing allograft survival timein a human wherein rejection of said allograft is mediated by rafkinase, said method comprising inhibiting human raf expression byadministering to said human an antisense oligonucleotide up to 50nucleotides in length and comprising SEQ ID NO: 1, wherein saidantisense oligonucleotide further comprises at least onephosphorothioate internucleoside linkage or at least one 2′ modificationof the sugar and wherein said antisense oligonucleotide inhibits humanraf gene expression.
 9. The method of claim 7 wherein said 2′modification is 2′—F, 2′—O—CH₃, 2′—O—CH₂CH₃ or 2′—O—CH₂CH₂CH₃.
 10. Themethod of claim 8 where said 2′ modification is 2′—F, 2′—O—CH₃,2′—O—CH₂CH₃ or 2′—O—CH₂CH₂CH₃.